Complete plastid genome of Gentiana zollingeri Fawcett (Gentianaceae) and phylogenetic analysis

Abstract Gentiana zollingeri Fawcett (Gentianaceae) belongs to the most species-rich section, Chondrophyllae, in the Gentianaceae, but its phylogenetic relationship with other members of this section is unclear. To confirm its phylogenetic position, the complete plastid genome of G. zollingeri was determined and analyzed. The plastome was sequenced using the Illumina HiSeq platform, assembled with GetOrganelle, and annotated with GeSeq. The genome is circular with a length of 130,762 bp. It contains a large single-copy (LSC) region of 74,236 bp, a small single-copy (SSC) region of 10,598 bp, and two inverted repeat (IR) regions of 22,964 bp each. The plastome of G. zollingeri shows considerable structural differences from those of other Gentiana plastomes, such as the absence of the ndh gene. In phylogenetic analyses, section Chondrophyllae, including G. zollingeri and its sisters, formed a long branch sistering with section Cruciata. The plastome sequence described here represents an important contribution to phylogenetic and evolutionary studies on Gentiana.

Gentiana is an alpine genus of about 360 species that are distributed worldwide, with the Qinghai-Tibet Plateau as the center of its distribution and diversity (Ho and Liu 2001;Favre et al. 2016). Out of the 13 sections in Gentiana, section Chondrophyllae Bunge is the most species-rich taxon, containing about 180 species (Ho and Liu 2001;Favre et al. 2020). Although many Gentiana plastome sequences are available, genome data are very limited for members of section Chondrophyllae (Fu et al. 2021). Gentiana zollingeri Fawcett, 1883 (Figure 1), having value in Chinese traditional medicine, belongs to section Chondrophyllae series Fastigiatae T.N. Ho, and is widely distributed in China, Russia, Korea, and Japan (Ho and Liu 2001).
Herein, we report the complete plastome of G. zollingeri (MZ934753) and its main characteristics. One G. zollingeri individual was collected from Liangjiatai Town, Hebei Province, China (40 46 0 N, 118 48 0 E). The voucher specimen was deposited at the Herbarium of Luoyang Normal University (Bin Cai, 987869364@qq.com) under the voucher number LP161491, and was identified by Dr. Pengcheng Fu. We extracted total DNA using a Dzup plant genomic DNA extraction kit (Sangon, Shanghai, China). The fragmented genomic DNA was sequenced using the Illumina HiSeq 2500 platform (Novogene, Tianjin, China), yielding 2 Gb clean data consisting of pairedend reads. The plastome was assembled using GetOrganelle version 1.7.1 (Jin et al. 2020) and annotated using GeSeq (Tillich et al. 2017) with the default parameters. The genome map was drawn using CPGview (http://www.1kmpg.cn/ cpgview) with the GenBank accession number (MZ934753).
The plastome was compared with those of species from the main clades of Gentiana Sun et al. 2018;Sun, Wang, et al. 2019;Fu et al. 2021), and structural changes were detected using mVISTA (Frazer et al. 2004). To confirm the phylogenetic position of G. zollingeri, the shared protein- Using concatenated data, maximum likelihood phylogenetic analyses were conducted using IQ-TREE version 1.6.12 (Nguyen et al. 2015) with 1000 replicates. The substitution model was chosen using ModelFinder 2 (Kalyaanamoorthy et al. 2017).
Metagentiana rhodantha (GenBank accession no. MN199153, Fu et al. 2021) was used as the outgroup. The complete plastome of G. zollingeri is 130,762 bp in length. The LSC and SSC regions consist of 74,236 and 10,598 bp, respectively, and there are two IRs of 22,964 bp each. A total of 122 genes are annotated, consisting of 80 protein-coding genes, 34 tRNA genes, and eight rRNA genes ( Figure 2). Comparative analyses indicate that the ndh complex in the plastome of G. zollingeri has undergone numerous gene loss (ndhA, ndhC, ndhE, ndhF, ndhG, ndhI, ndhJ and ndhK) and pseudogenization events (ndhB, ndhD and ndhH). The absence of the ndh complex is also detected in the Gentiana sections Chondrophyllae (Fu et al. 2021) and Kudoa (Sun et al. 2018).
The phylogenetic relationships among all main Gentiana sections are fully supported. The phylogenetic analysis shows that G. zollingeri is more closely related to G. aristata and G. producta than to G. leucomelaena, G. haynaldii, and G. cuneibarba (Figure 3), indicating that the series Humiles (containing G. aristata and G. leucomelaena) and Dolichocarpa (containing G. producta and G. haynaldii) in section Chondrophyllae are not monophyletic. Notably, G. filistyla forms a sister group with section Kudoa rather than with section Isomeria to which it belongs (Ho and Liu 2001). This finding is also supported by nuclear data (Favre et al. 2020). The plastome sequence of G. zollingeri reported here provides Graphic showing features of the Gentiana zollingeri plastome was generated using CPGview (http://www.1kmpg.cn/cpgview). The map contains seven circles. From center outwards, first circle shows distributed repeats connected with red (forward direction) and green (reverse direction) arcs. Second circle shows tandem repeats marked with short bars. Third circle shows microsatellite sequences as short bars.Fourth circle shows sizes of long single copy (LSC) and short single copy (SSC) regions. Fifth circle shows two inverted repeat (IR) regions: IRA and IRB. Sixth circle shows GC contents along plastome. Seventh circle shows genes marked with different colors according to their functional groups. new molecular data that illuminate aspects of the phylogenetic and molecular evolution of Gentiana, particularly the most species-rich section, Chondrophyllae.

Acknowledgments
We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. We also thank Rui Wang and Yizhuo Huang of Luoyang Normal University for their work in data analysis.

Ethical approval
The materials used in this study are not included in the IUCN Red List of Threatened Species, and the sampling site is not located in any protected area. The field study and laboratory research were conducted in accordance with the guidelines provided by Luoyang Normal University and Zhengzhou University.

Author contributions
J. Y. analyzed molecular data, prepare the illustrations and revised the manuscript. P. F. sampled individuals, prepared the illustration and wrote the draft; S. S. designed the study and revised the manuscript. All the authors read and approved the manuscript.

Disclosure statement
All the authors declare no conflicts in this study. The collection of the plant material in this study was carried out in accordance with the national and local regulations.

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at [https://www.ncbi.nlm.nih.gov] (https://www.ncbi.nlm.nih.gov/) under the accession no. MZ934753. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA758493, SRR15661475, and SAMN21028202, respectively.